This is a Target gene ORF that will be used to fuse with another carrier gene pr

Question

This is a Target gene ORF that will be used to fuse with another carrier gene present in a vector. Target gene ORF: S'ATG TCC CCC CGG AAA AAA GGG TGA 3' 3'TAC AGG GGG GCC ???? ??? ??? 5 Ncol 5'... C'CATGG...3 3'... GGTAcc...5 EcoR1 5'... G'AATTC...3 3'... CTTAAG...5 1-Give the primers needed to use in a PCR experiment using as template the DNA above, in order to add Ncol site by PCR to the left side of the Target DNA sequence above and an EcoRI to the right side, with some base changes, in order to obtain exactly the amplified DNA below (20pts) 5'C*C ATG GCC CCC CGG AAA AAA GGG TGA ATT C3' 3'G G TAC*CGG GGG GCC IIT ITT CCC ACT TAA*G5' Primers: 12 bases each: P1:........ P2.......... 2-Cut the product of question 1 with Ncol and EcoRI and give the central DNA product: (10pts)

Explanation / Answer

Q.1

5’ – C*CATG GCC CCC CGG AAA AAA GGG T*GA ATT C – 3’

3’ – G GTAC*CGG GGG GCC TTT TTT CCC A CT TAA*G – 5’

Forward Primer: 5’ - CCA TGG CCC CCC – 3’

Reverse Primer: 5’ – GAA TTC ACC CTT – 3’

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Q.2

5’ – C*CATG GCC CCC CGG AAA AAA GGG T*GA ATT C – 3’

3’ – G GTAC*CGG GGG GCC TTT TTT   CCC A CT TAA*G – 5’

5’ – *CATG GCC CCC CGG AAA AAA GGG T*                    – 3’

3’ –           *CGG GGG GCC TTT TTT CCC ACT TAA*       – 5’

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